It is estimated that 60-80% of the world's population is iron-deficient, making nutritional iron deficiency the most common nutritional disorder. Considerable evidence suggests that heme is an essential bioavailable iron source in human nutrition, but the pathways for heme absorption and utilization are poorly understood. Hemes also serve as prosthetic groups in proteins which play key roles in numerous and diverse biological processes. Since hemes are cytotoxic and insoluble, we hypothesize that specific pathways exist within cells for trafficking hemes from their site of synthesis or entry into the cell to various intracellular destinations. The long term goals of our lab are to identify the molecules and understand the mechanisms of heme uptake, utilization, transport, and storage in mammals. C. elegans cannot produce endogenous heme but depends on dietary heme to sustain metabolic processes. Like C. elegans, many parasitic nematodes including hookworms lack the heme biosynthetic pathway. Parasitic worms scavenge heme from their human host, which causes or exacerbates iron deficiency. C. elegans, therefore, represents a unique genetic animal model to identify heme transport pathways because they allow external control of heme levels not permissible in other organisms. We have synthesized a transgenic "heme-sensor" strain in C. elegans expressing the green fluorescent protein (GFP) under the control of a heme-responsive gene (hrg-1) promoter, which is sensitive to organismal heme levels. In our sensor strain, GFP fluorescence increases under low heme and decreases when the concentration of heme is elevated. We propose to use this strain in a functional reverse genetic screen using RNA-mediated interference. An E. coli mini feeding library, comprising ~400 heme responsive genes identified from DNA microarrays, will be used to expose worms to double-stranded RNA and "knock-down" target genes in the sensor strain. Changes in GFP fluorescence will be monitored as a function of heme levels and gene knock-downs. Clones that alter GFP intensity, as compared to controls, will be re-screened to eliminate false positives, and candidate genes identified from this screen will be confirmed by DNA sequencing. RNA extracted from worms grown in varying levels of heme and fed candidate clones will be characterized using quantitative real-time PCR and RNA blot analysis. The in vivo cellular localization of two such candidate proteins will be examined by microscopy;and their function(s) will be determined using fluorescent, radiolabeled, and toxic heme analogs. PUBLIC HEALTH RELEVANCE: In terms of public health relevance, results from the studies described in this proposal will provide new mechanistic insights into heme homeostasis in mammals. These insights may aid in the development of heme-based nutritional interventions for human iron deficiency and facilitate discovery of drugs that target heme transporters in parasitic worms, which exacerbate human iron deficiency